Metal ions such as Fe and Cu play an essential role in many cellular processes including energy production, biosynthesis and antioxidation. The key to their usefulness as enzyme cofactors lies in their ability to participate in electron transfer and to catalyze redox reactions. Yet this very chemistry imposes a stringent requirement to regulate the speciation, concentration and transport of cellular metal ions, since the free ions are themselves highly cytotoxic via Fenton mediated chemistry. Heme copper oxidases (HCOs) are classic examples of essential metalloproteins, and are utilized as the terminal electron acceptors in many prokaryotic and eukaryotic electron transfer chains. HCOs have evolved complex processes to assemble and metalate the multisubunit structures that are required for enzyme activity. In particular, the copper-binding Sco chaperone is required for the correct assembly and metalation of the mixed-valence dinuclear CuA center, which resides in subunit 2, and accepts electrons into the oxidase from cytochrome c. The chemistry at the heart of these cellular processes involves the exchange of CuI or CuII from chaperone to enzyme or transporter via molecular mechanisms which are only poorly understood. In this proposal we build on advanced spectroscopic methodologies developed in our laboratory to unravel the detailed mechanisms of metal transport and exchange. These experiments are underpinned by our expertise in X-ray absorption spectroscopy which is the only spectroscopic technique capable of directly observing the CuI state as it is transferred along the homeostatic pathways. Specifically we propose to apply the technique of Se labeling coupled to XAS detection to investigate the metallation of the CuA center by its putative chaperone Sco and by the periplasmic copper-binding protein PCuAC or its homologues. Spectroscopic and kinetic studies of metal transfer will be correlated with function across a number of different prokaryotic and eukaryotic systems, using relationships between mutagenesis and phenotype to elucidate the structural determinants of the transfer mechanism. These studies will lead to a better understanding of the molecular basis of copper trafficking and will aid in combating diseases of aberrant copper homeostasis.